PROJECT 1 - SUMMARY Background: Mortality of Clostridium sordellii, C. novyi and C. difficile infections is highly correlated with the development of a leukemoid reaction (LR), yet the mechanisms driving the LR have not been elucidated. Each of these organisms produces large clostridial cytotoxins (LCCs), suggesting their role in LR development. However, LR intensity is species-specific with that of C. sordellii being extreme. Hypothesis: We hypothesize that the shared LCCs compromise the bone marrow sinusoidal-endothelial barrier, resulting in the premature egress of granulocytes into circulation and that C. sordellii-specific exotoxins significantly augment this process. In support of this latter notion, we have identified a unique C. sordellii metalloproteinase we call Mcs1 that specifically cleaves key adhesins that tether hematopoietic precursors to the bone marrow stroma. The current study will investigate the roles of LCCs and Mcs1 in driving the common and the extreme LRs using relevant C. difficile and C. sordellii infection models. Specific Aim 1: To determine the role of LCCs in mediating the common clostridial LR. Mice will be infected with wild-type or LCC-deficient C. difficile or C. sordellii, in the presence or absence of a neutralizing anti-LCC antibody. Having implicated a role for neutrophil elastase in this process, separate groups of infected mice will receive an elastase inhibitor. Survival, LR development, and expression of phagocyte maturity markers will be followed. Specific Aim 2: To establish the in vivo role of the C. sordellii Mcs1 in augmenting the clostridial LR. rMcs-1 has been generated and will be used to actively immunize mice that are subsequently challenged with WT C. sordellii. An Mcs1- deficient mutant will be developed and studied in C. sordellii infection. In both cases, mortality, LR development and histopathology of infected tissues will be followed. Specific Aim 3: To define how C. sordellii infection influences the bone marrow endothelium. The bone marrow ultrastructure and expression of adherence molecules regulating granulopoiesis in response to C. sordellii infection with WT organisms or LCC- or Mcs-1 mutants will be evaluated utilizing TEM and immunohistochemistry. This aim will heavily utilize the HPIC facility. Specific Aim 4: To assess the in vitro and in vivo effects of C. sordellii and C. difficile cytotoxins on neutrophil trafficking. Neutrophil transmigration will be assessed in vivo during active C. difficile and C. sordellii infection using our live animal IVIS imaging system and transgenic mice expressing fluorescent protein on their neutrophil surface. Impact on Human Health: Understanding the mechanisms driving the LR may lead to novel forms of prevention and treatment for these devastating infections and costly infections. Contribution to Multi-disciplinary Infectious Diseases Research Program: The PI is a respected scientist in clostridial pathogenesis and will bring expertise in anaerobic infections to the ID program. His in-depth cellular and molecular knowledge will increase the core's understanding of the effects of bacterial toxins on hematopoiesis and the inflammatory response.